Cell membrane-camouflaged liposomes and neopeptide-loaded liposomes with TLR agonist R848 provides a prime and boost strategy for efficient personalized cancer vaccine therapy.

Recent advances in bioinformatics and nanotechnology offer great opportunities for personalized cancer vaccine development. However, the timely identification of neoantigens and unsatisfactory efficacy of therapeutic cancer vaccines remain two obstacles for clinical transformation. We propose a "prime and boost" strategy to facilitate neoantigen-based immunotherapy. To prime the immune system, we first constructed personalized liposomes with cancer cell membranes and adjuvant R848 to provide immunostimulatory efficacy and time for identifying tumor antigens. Liposomes loaded with personalized neopeptides and adjuvants were used to boost the immune response. In vitro experiments verified potent immune responses, including macrophage polarization, dendritic cell maturation, and T lymphocyte activation. In vivo B16F10 and TC-1 cancer model were used to investigate efficient tumor growth suppression. Liposomal vaccines with neopeptides could stimulate human dendritic cells and T lymphocytes in vitro. These results demonstrate that the "prime and boost" strategy provides simple, quick, and efficient personalized vaccines for cancer therapy.

Ultrasensitive ELISA using enzyme-loaded nanospherical brushes as labels.

Improving the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is of utmost importance for meeting the demand of early disease diagnosis. Herein we report an ultrasensitive ELISA system using horseradish peroxidase (HRP)-loaded nanospherical poly(acrylic acid) brushes (SPAABs) as labels. HRP was covalently immobilized in SPAABs with high capacity and activity via an efficient "chemical conjugation after electrostatic entrapment" (CCEE) process, thus endowing SPAABs with high amplification capability as labels. The periphery of SPAAB-HRP was further utilized to bind a layer of antibody with high density for efficient capture of analytes owing to the three-dimensional architecture of SPAABs. Using human chorionic gonadotrophin (hCG) as a model analyte, the SPAAB-amplified system drastically boosted the detection limit of ELISA to 0.012 mIU mL(-1), a 267-fold improvement as compared to conventional ELISA systems.

Covalent immobilization of proteins on 3D poly(acrylic acid) brushes: mechanism study and a more effective and controllable process.

Polymeric brushes have emerged as a novel 3D material platform that provides great amounts of binding sites for biomolecules. This paper investigates the covalent immobilization mechanism of protein by spherical poly(acrylic acid) brushes (SPAABs) in the widely adopted N-hydroxysuccinimide/N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimide hydrochloride (NHS/EDC) process. It was discovered that electrostatic interaction plays a crucial role in the covalent immobilization of protein. Due to the existence of 3D architecture and "Donnan effect", SPAABs exhibit quite different immobilization kinetics in comparison with conventional 2D materials. Under conditions favorable to electrostatic interaction, the effect of "electrostatic interaction induced covalent binding" was observed as a result of competitive immobilization by physical adsorption and chemical binding. On the basis of the mechanism study, a new "chemical conjugation after electrostatic entrapment" (CCEE) method was developed which set the chemical and physical immobilization process apart. A more effective and well-defined covalent immobilization was achieved. And the binding capacity can be tuned in a wide range (0-4.2 mg protein/mg SPAABs) with a high level of control.

A new method for the determination of critical polyethylene glycol concentration for selective precipitation of DNA fragments.

Separation strategies based on size-selective precipitation of DNA fragments with polyethylene glycol (PEG) have been used for achieving desired DNA interval in automated sample preparation for next-generation sequencing. By varying PEG concentration, DNA fragments of different sizes can be precipitated onto surfaces of carboxyl-coated paramagnetic particles selectively, and therefore, the desired DNA interval can be obtained. However, one of the crucial points in this approach is to determine the critical PEG concentration for DNA fragment of a certain size. The aim of this work was to develop a convenient and reliable method for accurately determining the critical PEG concentration. In our method, at a fixed concentration of sodium chloride (NaCl), recovered DNA samples obtained with different PEG concentrations were directly quantified, and their concentrations as a function of the PEG concentration were fitted by the logistic function. The critical PEG value was easily and accurately determined from the fitted logistic function. The repeatability and stability of the critical PEG value were assessed, showing an excellent reliability of the method. Based on this method, critical PEG values of different-size DNA fragments were determined at different NaCl concentrations. The effectiveness of the method was also demonstrated by selective precipitation of DNA fragments.

Magnetite nanocrystal clusters with ultra-high sensitivity in magnetic resonance imaging.

Magnetic iron oxide particles are widely used as contrast agents to improve the sensitivity of magnetic resonance imaging (MRI). Their efficiency in MRI is usually quantified by transverse relaxivity (r(2)) in solution. Herein, we synthesized a series of magnetite nanocrystal clusters (MNCs) with ultra-high transverse relaxivity by a polyol process and studied the relationship between r(2) and size of the MNCs. The sizes of MNCs can be tuned over a wide range from 13 to 179 nm. The r(2) of MNC suspensions as a function of the size of the cluster was analyzed and compared with a theoretical model. We found that MNCs of 64 nm had an r(2) value of 650 mM(-1) s(-1), which was more than three times that of the commercial contrast agent and was among the highest reported for iron oxide materials. Compared with the theoretical model, the r(2) value of the MNC suspension is approximately 0.93 of the theoretical prediction. Imaging of the MNC suspensions was performed in a clinical 1.5 T MRI instrument and a comparison was made between MNCs and commercial contrast agents. MRI indicated that the decrease of signal intensity induced by MNCs was in proportion to the r(2) value, which was in accordance with theoretical predictions. These results demonstrate that MNCs with ultra-high transverse relaxivity and tunable size are promising candidates for molecular imaging and clinical diagnosis in MRI.

A facile polymer mediated dye incorporation method for fluorescence encoded microbeads with large encoding capacities.

Here we report a facile dye incorporation method for fluorescence encoded microbeads, which is achieved by tuning the mixed polymer type (blank and dye-labeled polymers) and their doping ratio through electrostatic loading into mesoporous beads. This method is universal to various carriers and could render large encoding capacities.

In situ preparation of magnetic nonviral gene vectors and magnetofection in vitro.

Magnetic nonviral gene vectors were in situ prepared in the presence of ferrous salts and hyperbranched poly(ethylenimine)s (HPEI) with different molecular weights. HPEI, one of the most promising nonviral vectors, was not only utilized as the nanoreactor and stabilizer to prepare magnetic nanoparticles, but also skillfully used as a base supplier to avoid introducing alkali hydroxide or ammonia. Magnetic nonviral gene vectors with various magnetite contents and saturation magnetizations were obtained by changing the weight ratio of HPEI to FeSO(4).7H(2)O and the molecular weight of HPEI. MTT assays suggested that the resulting magnetite/HPEI gene vectors had lower cytotoxicity compared with pure HPEI. The magnetite/HPEI nonviral gene vectors were used for magnetofection. It was found that the luciferase expression level mediated by magnetite/HPEI in COS-7 cells under a magnetic gradient field was approximately 13-fold greater than that of standard HPEI transfection.

Superparamagnetic magnetite nanocrystal clusters: a sensitive tool for MR cellular imaging.

Silica coated, PEI and citric acid hybrid superparamagnetic magnetite nanocrystal clusters (SMNC) were synthesized using either a mini-emulsion/sol-gel method or a polyol technique. After careful characterization of the size, structure, composition, and magnetic properties, the as-synthesized SMNC were used for cell labeling while the MR detection sensitivity of cells labeled with silica SMNC was performed with a 3 T whole body MR scanner. TEM investigations revealed that the sizes of the SMNC were about 200 nm and the SMNC mainly consisted of magnetite nanoparticles imbedded in a PEI, citric acid or polystyrene scaffold. Silica and citric acid SMNC were highly negatively charged and PEI SMNC were positively charged. Relaxometry measurements revealed that these SMNC possessed a very high MR sensitivity (silica SMNC: r(2) = 299 s(-1) mM(-1), PEI SMNC: r(2) = 124 s(-1) mM(-1)), especially for the citric acid SMNC (r(2) = 360 s(-1) mM(-1)). Furthermore, when used for cell (RAW264.7 cells) labeling, the SMNC had no adverse effect on cell viability, and the cell uptake of the SMNC show a dose- and time-dependent feature. MR imaging of cells labeled with silica SMNC indicated that cells with a concentration as low as 10 x 10(3) cells ml(-1) could be detected with a 3 T MRI scanner. Our study demonstrated that superparamagnetic magnetite nanocrystal clusters are a sensitive tool for cell imaging.

A T1/T2 dual functional iron oxide MRI contrast agent with super stability and low hypersensitivity benefited by ultrahigh carboxyl group density.

Clinically acceptable safety and efficacy are the most important issues for the design and synthesis of iron oxide MRI contrast agents. In order to meet the practical requirements, a kind of low molecular weight PAA-coated Fe3O4 nanoparticle (CS015) with super colloidal stability and low hypersensitivity benefitting from an ultrahigh carboxyl group density was developed in this study. The composition and physicochemical properties of the particles were characterized by TEM, XRD, FTIR and TGA. The ultrahigh density of COOH on the particles (33 COOH per nm2) was verified while a core size of 5.1 nm and a dynamic diameter of 41 nm with a narrow distribution were also achieved. The particles still showed excellent dispersity and stability even after a spray-drying or freeze-drying process, exposure to high temperature sterilized conditions and long-term storage. The nanoparticles could quickly capture iron ions in bulk solution which was confirmed by ITC results, and the bioactive iron concentration of CS015 was greatly decreased (0.54 ± 0.05 mg L-1) compared to that of commercially available ferumoxytol, iron sucrose and VSOP. Free iron ion release was 1120 times lower than the toxic concentration of iron. An excellent biocompatibility of CS015 with no obvious cytotoxicity and low risk of hypersensitivity has been manifested by cytotoxicity experiments and a passive cutaneous anaphylaxis test. The T1 and T2-weighted MRI contrast effects both in vitro and in vivo have also been verified which made CS015 a potential dual MRI contrast agent. Furthermore, theoretically calculated conformation was speculated and all the advantages mentioned above were benefited from the three dimensional brush-like texture of CS015. Therefore, these merits make the CS015 nanoplatform highly suitable in diagnostic applications as a MRI contrast agent.

Strategy to prevent cardiac toxicity induced by polyacrylic acid decorated iron MRI contrast agent and investigation of its mechanism.

Polyelectrolyte modified iron oxide nanoparticles have great potential applications for clinical magnetic resonance imaging (MRI) and anemia treatments, however, possible associated heart toxicity is rarely reported. Here, polyacrylic acid (PAA)-coated Fe3O4 nanoparticles (PION) were synthesized and lethal reactions appeared when it was applied in vivo. The investigation of underlying mechanism showed that PION could break electrolyte balance and further resulted in serious heart failure, which was observed under color doppler ultrasound and dynamic vector blood flow technique. The results demonstrated that PION had a strong absorption tendency for divalent ions and the maximum tolerated dose (MTD) was lower than 100 mg/kg. From electrocardiography (ECG), PION presented an obvious impact on CaV1.2 ion channel, which leading to fatal arrhythmia. An appropriate solution for preventing this deadly effect was pre-chelation Ca2+ (n (Ca): n (COOH) = 3: 8) to PION (PION-Ca), which displayed much higher cardiac and electrophysiological safety when sealing the binding point of divalent cation ions with PAA. The injection in Beagle dogs further confirmed the safety of PION-Ca. This study explored the mechanism and offered a solution for cardiac toxicity induced by PAA-coated nanoparticles, which guides for enhancing the safety of such polyelectrolyte decorated nanoparticles and provides assurance for clinical applications.

Design and preparation of bi-functionalized short-chain modified zwitterionic nanoparticles.

An ideal nanomaterial for use in the bio-medical field should have a distinctive surface capable of effectively preventing nonspecific protein adsorption and identifying target bio-molecules. Recently, the short-chain zwitterion strategy has been suggested as a simple and novel approach to create outstanding anti-fouling surfaces. In this paper, the carboxyl end group of short-chain zwitterion-coated silica nanoparticles (SiO2-ZWS) was found to be difficult to functionalize via a conventional EDC/NHS strategy due to its rapid hydrolysis side-reactions. Hence, a series of bi-functionalized silica nanoparticles (SiO2-ZWS/COOH) were designed and prepared by controlling the molar ratio of 3-aminopropyltriethoxysilane (APTES) to short-chain zwitterionic organosiloxane (ZWS) in order to achieve above goal. The synthesized SiO2-ZWS/COOH had similar excellent anti-fouling properties compared with SiO2-ZWS, even in 50% fetal bovine serum characterized by DLS and turbidimetric titration. Subsequently, SiO2-ZWS/COOH5/1 was chosen as a representative and then demonstrated higher detection signal intensity and more superior signal-to-noise ratios compare with the pure SiO2-COOH when they were used as a bio-carrier for chemiluminescence enzyme immunoassay (CLEIA). These unique bi-functionalized silica nanoparticles have many potential applications in the diagnostic and therapeutic fields. STATEMENT OF SIGNIFICANCE: Reducing nonspecific protein adsorption and enhancing the immobilized efficiency of specific bio-probes are two of the most important issues for bio-carriers, particularly for a nanoparticle based bio-carrier. Herein, we designed and prepared a bi-functional nanoparticle with anti-fouling property and bio conjugation capacity for further bioassay by improving the short-chain zwitterionic modification strategy we have proposed previously. The heterogeneous surface of this nanoparticle showed effective anti-fouling properties both in model protein solutions and fetal bovine serum (FBS). The modified nanoparticles can also be successfully functionalized with a specific antibody for CLEIA assay with a prominent bio-detection performance even in 50% FBS. In this paper, we also investigated an unexpectedly fast hydrolysis behavior of NHS-activated carboxylic groups within the pure short-chain zwitterionic molecule that led to no protein binding in the short-chain zwitterion modified nanoparticle. Our findings pave a new way for the designing of high performance bio-carriers, demonstrating their strong potential as a robust platform for diagnosis and therapy.

Levofloxacin loaded mesoporous silica microspheres/nano-hydroxyapatite/polyurethane composite scaffold for the treatment of chronic osteomyelitis with bone defects.

Chronic osteomyelitis is a prolonged persistent disease accompanied by bone destruction and sequestrum formation, it is very difficult to treat. Antibiotic loaded polymethyl methacrylate (PMMA) has been used in clinical. However, when PMMA was implanted in the body, the deficiencies is that it is non-biodegradable and a second operation is needed. Here, we synthesize a novel levofloxacin loaded mesoporous silica microspheres/nano-hydroxyapatite/polyurethane composite scaffolds, and evaluated the therapeutic effect in treating chronic osteomyelitis with bone defects in rabbit model compared with bulk PMMA. X-ray, Micro CT, gross pathology as well as immunohistochemical staining were performed at predesignated time points (1, 3, 6 and 12 weeks). Our results demonstrated that the efficiency of mesoporous silica microspheres/nano-hydroxyapatite/polyurethane composite scaffolds loaded with 5 mg levofloxacin was much better at treating bone defects than the other groups. This novel synthetic scaffold may provide a solution for the treatment of chronic osteomyelitis.

[Security evaluation of subcutaneous injection with water-based dextran-coated magnetic fluid].

Water-based magnetic fluid was synthesized by using 50% dextran 40,000 as coated reagent. The acute toxicity and irritant of the magnetic fluid injected into mice subcutaneous tissues were examined. The lethal dosage 50 of dextran-coated magnetic fluid was 4409.61 +/- 514.93 mg/kg. Twenty four h after subcutaneous injecting with 30 mg/0.3 ml dextran-coated magnetic fluid, no more inflammation than hemangiectasia and leucocytes infiltration had been seen in subcutaneous tissues, 72 h later the reaction phenomena disappeared. While, injection with 30 mg/0.3 ml water-based oleate sodium-coated magnetic fluid, ulceration and break-off of cutis had been seen in the seventh days. That is to say, the dextran-coated magnetic fluid was safe and well tolerate, however, the oleate sodium-coated magnetic fluid was not fit to subcutaneous injection.

Synthesis and Biomedical Applications of Poly((meth)acrylic acid) Brushes.

Poly((meth)acrylic acid) (P(M)AA) brushes possess a number of distinctive properties that are particularly attractive for biomedical applications. This minireview summarizes recent advances in the synthesis and biomedical applications of P(M)AA brushes and brushes containing P(M)AA segments. First, we review different surface-initiated polymerization (SIP) methods, with a focus on recent progress in the surface-initiated controlled/living radical polymerization (SI-CLRP) techniques used to generate P(M)AA brushes with a tailored structure. Next, we discuss biomolecule immobilization methods for P(M)AA brushes, including physical adsorption, covalent binding, and affinity interactions. Finally, typical biomedical applications of P(M)AA brushes are reviewed, and their performance is discussed based on their unique properties. We conclude that P(M)AA brushes are promising biomaterials, and more potential biomedical applications are expected to emerge with the further development of synthetic techniques and increased understanding of their interactions with biological systems.

Functional short-chain zwitterion coated silica nanoparticles with antifouling property in protein solutions.

A new functional nanoparticle, consisting of a silica core onto which short-chain zwitterions are chemically connected, was successfully prepared and showed excellent antifouling performance to protein solutions. These nanoparticles (NPs) own excellent stability even in 1M NaCl solutions for at least 48 h. The interaction between these "zwitterated" NPs and proteins were investigated by dynamic light scattering (DLS), turbidimetric titration, and isothermal titration calorimetry (ITC). The results demonstrated that the zwitterated NPs had antifouling property both in single protein solutions and serum (fetal bovine serum, FBS). The zwitterated NPs also own abundant functional groups which could conjugate with biomolecules for future applications in therapeutic and diagnostic field.

Application of hydrophobic coatings in biodegradable devices.

BACKGROUND: Degradation periods of biodegradable medical devices strongly affect their clinical performance and therefore special attention has been drawn to modulate their degradation rate. OBJECTIVE: This paper presents an experimental study on the effect of hydrophobic coating on the degradation behavior of PLLA samples. METHODS: PLLA films were coated with a thin layer of PCL, and a combination of scanning electron microscopy (SEM), Ubbelohde Viscometer Capillary, and chromatograph of gel permeation (GPC) was used to evaluate the morphology and molecular weight changes of samples during degradation. In addition, the mass loss of samples was also measured during the experiment. RESULTS: PLLA samples with PCL coatings showed a slower degradation rate than those without PCL coatings, which indicated that PCL coatings could protect inside PLLA samples and slow down the degradation rate of PLLA samples. CONCLUSIONS: The results of the study suggest that hydrophobic coating on polymer materials is a useful approach to control the degradation of polymer medical device.

Improvement of Protein Immobilization and Bioactivity of Magnetic Carriers Using a Brushed Beads-on-Beads Structure.

To achieve higher protein immobilization and bioactivity, as well as automatic manipulation, we prepared a new type of biocarrier based on the brushed beads-on-beads structure. Many poly(acrylic acid) (PAA) brushed nanoparticles were packed onto the surface of amino-functionalized magnetic particles through an efficient carbodiimide-assisted coupling reaction to attain a hierarchical structure, a unique three-dimensional (3D) space and automatic manipulation characteristics. The proposed biocarrier was evaluated in the recognition capability of the immunocomplex and showed a 6.7-fold increase compared with control beads with a hard surface. The results of this study suggest promising applications in targeted capture and high-performance biodetection processes.

Low toxicity and long circulation time of polyampholyte-coated magnetic nanoparticles for blood pool contrast agents.

Polyampholyte-coated (poly(acrylic acid) (PAA)-co-3-(diethylamino)-propylamine (DEAPA)) magnetite nanoparticles (PAMNPs) have been prepared as contrasting agent used in magnetic resonance imaging (MRI). Excellent biocompatibility is required for contrasting agents used in high-resolution magnetic resonance angiography. To evaluate the biocompatibility of PAMNPs, some experiments have been conducted. The hemolysis, plasma recalcification, dynamic blood clotting, prothrombin time, inflammatory cytokine release and complement system activation assays were carried out to investigate the hemocompatibility. To evaluate the toxicity to vessel, MTT test and vascular irritation tests were conducted. Tissue toxicity test was also performed to investigate the biocompability in vivo. We also looked into the biodistribution. The results showed that PAMNPs at the working concentration (0.138 mM) present similar hemocompatibility with negative control, thus have no significant effect to vessels. PAMNPs were mainly distributed in the liver and the blood. The circulation time in blood was considerably long, with the half-time of 3.77 h in plasma. This property is advantageous for PAMNPs' use in angiography. PAMNPs could be metabolized rapidly in mice and were not observed to cause any toxic or adverse effect. In short, these results suggest that the PAMNPs have great potential to serve as safe contrast agents in magnetic resonance imaging (MRI).

Size-selective DNA separation: recovery spectra help determine the sodium chloride (NaCl) and polyethylene glycol (PEG) concentrations required.

In the presence of sodium chloride (NaCl), DNA fragments can be size-selectively separated by varying the final concentration of polyethylene glycol (PEG). This separation strategy in combination with the use of paramagnetic particles provides a valuable platform for achieving the desired DNA size interval, which is important in automated library preparation for high-throughput DNA sequencing. Here, we report the establishment of recovery spectra of DNA fragments that enable the determination of suitable NaCl and PEG concentrations for size-selective separation. Firstly, at a given NaCl concentration, the recovery equation was obtained by fitting the DNA recovery ratios versus the PEG concentrations using the logistic function to determine the required parameters. Secondly, the slope function of the recovery equation was achieved by deducing its first derivative. Therefore, the recovery spectrum can be generated using the slope function based on those parameters. According to the recovery spectra of different length DNA fragments, suitable NaCl and PEG concentrations can be determined, respectively, by calculating their resolution values and recovery ratios. The strategy was effectively applied to the size-selective separation of 532-, 400-, and 307-bp fragments at the selected reagent concentrations with recoveries of 96.9, 64.7, and 85.9%, respectively. Our method enables good predictions of NaCl and PEG concentrations for size-selective DNA separation.